Morphogenetic identification of a new record Deudorix livia (Lepidoptera: Lycaenidae) in Assiut Governorate of Northern Upper Egypt

Deudorix livia (Klug, 1834) (Lepidoptera: Lycaenidae) is one of the most serious lepidopteran insect pests attacking pomegranate fruit around the world, including Egypt (Assiut Governorate, Upper Egypt). To create an effective program (IPM) to control such harmful pests, accurate identification of the pest morphology and genetic structure is essential. Studies on the morphogenetics of this pest are scarce. So, the goal of this research is to identify it both morphologically and genetically. Pomegranate butterfly immature stages were collected from infested pomegranate fruits and reared in the laboratory until the adult's emergence. By using light and scanning electron microscopy, some morphological structures of males and females were studied. DNA was extracted from the legs of a pomegranate butterfly adult. Also, PCR was conducted by using the mitochondrial CO1 gene for sequencing and phylogenetic tests. The results show that the body scales are a mixture of dark and light gray on the dorsal side and white on the ventral side in both sexes. The average male body length (BL) was 11.674 ± 0.299 mm and was 11.458 ± 1.001 mm for the females. The wing venation is similar in both sexes. For the first time, a partial sequence of the mitochondrial CO1 gene in D. livia was deposited in GenBank (MW463927).


Morphogenetic identification of a new record Deudorix livia (Lepidoptera: Lycaenidae) in Assiut Governorate of Northern Upper Egypt
Deudorix livia (Klug, 1834) (Lepidoptera: Lycaenidae) is one of the most serious lepidopteran insect pests attacking pomegranate fruit around the world, including Egypt (Assiut Governorate, Upper Egypt).To create an effective program (IPM) to control such harmful pests, accurate identification of the pest morphology and genetic structure is essential.Studies on the morphogenetics of this pest are scarce.So, the goal of this research is to identify it both morphologically and genetically.Pomegranate butterfly immature stages were collected from infested pomegranate fruits and reared in the laboratory until the adult's emergence.By using light and scanning electron microscopy, some morphological structures of males and females were studied.DNA was extracted from the legs of a pomegranate butterfly adult.Also, PCR was conducted by using the mitochondrial CO1 gene for sequencing and phylogenetic tests.The results show that the body scales are a mixture of dark and light gray on the dorsal side and white on the ventral side in both sexes.The average male body length (BL) was 11.674 ± 0.299 mm and was 11.458 ± 1.001 mm for the females.The wing venation is similar in both sexes.For the first time, a partial sequence of the mitochondrial CO1 gene in D. livia was deposited in GenBank (MW463927).

Morphological identification
Body coloration, wing venation, leg structure, and body morphometrics are the morphological criteria used for identifying pomegranate butterfly 9,10 .

Scanning electron microscopy (SEM)
Skeletal structures were studied using a Jeol JSM-5500.LV(JEOL-Japan) scanning electron microscope in The Central Lab., Fac. of Sci., South Valley Univ.

Light microscope
Wing scales were removed according to Belkin 11 , and the bleached wings were mounted on slides for studying wing venation by using the Leica DC150 Camera.

Figure 1.
Immature stages of pomegranate butterfly (eggs and larvae) collected from infested fruits; (a) a red circle surrounds 1st larval instar hatched from the egg, and (b) a black circle surrounds the 4th larval instar with symptoms of infestation caused by larvae that inter-feeding fruits.

Body morphometrics
The criteria of measurements were body length (BL = from the head to the tip of the abdomen (Fig. 2a)), fore wing length (FWL = from the base (humeral angle) to the apex (apical angle) (Fig. 2b)), hind wing length (HWL = from the base to the middle of the term (apical margin), (Fig. 2c)), and antennal length (AntL = from the base to the tip of the antenna (Fig. 2d)) 10 .Measurements determined by the HDMI MULTI-OUTPUT HD' (Toup Cam_120) CAMERA.

Molecular genetic identification of pomegranate butterfly
DNA isolation DNA was extracted from the specimen's legs from a pomegranate butterfly and followed by a modified cetyltrimethylammonium bromide protocol with an additional polyethylene glycol precipitation as described by Reineke 12 .

Polymerase chain reaction (PCR) conditions
The mitochondrial CO1 gene was amplified using the primers LepF1 (ATT CAA CCA ATC ATA AAG ATA TTG G) and LepR1 (TAA ACT TCT GGA TGTCC-AAA AAA TCA) 7 .The PCR reactions comprised 1μL (10 pmol) of each forward and reverse primer, 1μL of genomic DNA, and 20μL PCR master mix in a final reaction volume of 40μL.The PCR was carried out under the following conditions: denaturing at 95 °C for 4 min., followed by 34cycles of denaturing at 94 °C for 60 s., alignment at 48 °C for 60 s., and extension at 72 °C for 60 s., finishing with an extension at 72 °C for 7 min.All PCR products were visualized using 1.5% agarose gel stained with ethidium bromide.Gel electrophoresis was run for 40 min., at 100 V using 100 bp DNA Ladder RTU (Ready-to-Use) GeneDireX.

Sequence and phylogenetic analysis
All DNA sequencing was achieved by Macrogen (Seoul, South Korea).The sequences were deposited at the National Centre for Biotechnology Information (GenBank/NCBI) to obtain the accession number.DNA sequences were primarily aligned with the default parameters of CLUSTALW 13 .Two methodologies, Minimum Evolution (ME) and Neighbour-joining (NJ) implemented in MEGA software version 7.0 18 14 , were used for phylogenetic reconstructions.In this study, 1000 bootstrap iterations 15 were applied.Sequence divergences were calculated using Kimura 2-parameter distances 16 .

GenBank accession number
The partial sequence of the CO1 region of the butterfly reported in this paper is deposited in GenBank nucleotide sequence databases (http:// www.ncbi.nlm.nih.gov) under accession number MW463927 for DAUF-Pomegranate.

Data analysis
Means ± standard deviation (SD) was determined by using Microsoft Excel 2016 for morphometric measurement data.

Morphological identification
Using light microscopy and scanning electron microscopy (SEM), some morphological structures of male and female pomegranate butterfly were illustrated.

The general body coloration
The general color of body scales is a mix of dark and light grey on the dorsal side and white on the ventral side in both sexes (Fig. 3).
The head Compound eyes.The color of compound eyes was brown and coppery, (Fig. 4a,b).For the first time study by focusing on one scanned eye, it there are the interfacial hairs (Fig. 4c,d) were found distributed across the eye in a corner between three facets.Antennae.The antenna of the pomegranate butterfly is clavate type (Fig. 5).The number of segments in the flagellum is 22 filiform segments followed by 17 clubbed ones in both sexes.The color of terminal segments is orange and black, but other segments are black and white.
Mouthparts.Mouthparts of pomegranate butterfly are sucking type.There are a pair of three-segmented labial palps and a brown coiled proboscis (galea).The labial palps tip is covered with brown scales and the bases with white scales (Fig. 6).www.nature.com/scientificreports/ The thorax In both sexes, the coloration of the thorax is shiny dark grey, and the smallest of the three thoracic segments is the prothorax in a pomegranate butterfly.

Legs
The legs of a pomegranate butterfly are walking type.The three pairs of legs are covered with white scales.Each leg consists of a coxa, trochanter, femur, tibia, and five segmented fused tarsi (Fig. 7a).Tibiae of legs apically bear well-developed branched spurs.The apical tarsomere bears two claws with pulvilli and empodium in all legs for both sexes except the foreleg of the male consists of one claw (Fig. 7b,c).

Wings
Wing coloration.Both sexes of pomegranate butterfly differed in their coloration.
Upper side of female and male wing.The female fore wing upper side scaled dark brown in the apical angle (apical and sub-apical area) and apical margin (marginal and sub-marginal area), brown color in the post-discal and discal area, and a bluish tint becoming darker in the humeral angle (basal and post-basal area) (Fig. 8a FW).The female hind wing's upper side is covered in brown scales except for the anal margin (tornal area) and humeral angle (part of the discal, sub-discal, basal, and dorsal areas), which are squirrel color.Also, two spotted patches were recognized nearby the anal angle (tornal area), (Fig. 8a HW).
The upper side of the male forewing is covered by dark orang scales, except for the costal margin (costal area), apical angle (apical and subapical area), and apical margin (marginal area), which are brown.Except for the humeral angle (basal area), anal margin (dorsal area), and part of (sub-discal, discal, and post-discal area) (Fig. 8b FW).The male hind wing's upper side is covered by dark orange scales.Additionally, there was one spotted patch in the sub-discal area and near the humeral angle (basal area) (Fig. 8b HW).
Lower side of female and male wing.The lower sides of the wings are similar in both sexes (Fig. 9).The fore and hind wings' lower sides are covered with grey scales with different scattered bands.There are two black rounded spots, one just above the filamentous tail and the other on the wing tip.It is worth pointing out herein that in the sub-discal area of the hindwing lower side, clearly encounter two dark spots surrounded by a white oval circle (Fig. 9a,b HW).

Morphometrics of pomegranate butterfly
Body length (BL) measurements.The (BL) in both sexes was measured for each sex in the pomegranate butterfly (Table 1).In males, (BL) ranged from 11.360 to 12.000 mm with an average of 11.674 ± 0.299 mm, and in females ranged from 10.380 to 13.090 mm with an average of 11.458 ± 1.001 mm.
Antennal length.The distance from the base to the tip of the antenna was measured for antennal length.The antennal length measurement of the pomegranate butterfly ranged from 6.190 to 7.850 mm with an average of 7.203 ± 0.529 mm.The above-mentioned measurements are based on ten individuals (n = 10).

Molecular identification of pomegranate butterfly
The first-time record of Deudorix livia, in the GenBank for the partial nucleotide sequence of CO1 was deposited under the accession number MW463927.The sequencing of the mitochondrial CO1 gene produced a nucleotide length of 632 bp.The nucleotide frequencies of adenine (A), thymine (T), cytosine (C), and guanine (G) were 33.7, 37.8, 14.8, and 13.7%, respectively.The average A + T content was 71.5%, which was higher than the average C + G content (Table 3).
The sequences of CO1 of D. livia were subjected to BLAST/N at (NCBI) and revealed eight related species from the genus Deudorix; in addition to the out-group species; Sabatinca demissa (HM431779.1),Sabatinca calliarcha (HM431781.1)and Sabatinca heighwayi (HQ575067.1) of the family Micropterigidae (Table 4).www.nature.com/scientificreports/Among the studied D. livia species pairwise genetic distances ranged from 0.0114 to 0.0151.The most related species to our sample was Deudorix isocrates, where the genetic distance was 0.0114.The most genetic distance to our sample was Deudorix epirus, where the genetic distance was 0.0141.Overall, the mean distance value was 0.17% (Table 5).
To conduct the phylogenetic tree analysis using CO1 sequencing, D. livia was submitted to be analyzed together with eight related Deudorix species sequences and the out-group species from GenBank/NCBI (previously mentioned in Table 5).For more illustrative phylogenetic relations, more than one phylogenetic method was used (ME and NJ) based on the CO1 gene.These methods showed nearly the same relations with some differences in support values and revealed 3 main features: (1) species of out-group formed a separate cluster.(2) all Deudorix species formed two main clades; the first includes D. smilis and D. democles while the second contains the rest species.(3) the most related species to D. livia crops was D. isocrates (Fig. 12a,b).

Discussions
The Lycaenidae is a family comprising several thousand species.Many are brightly colored, and this is often the result of interference effects caused by the microstructure of the wings.One of the genera in this family is the Deudorix genus.This genus is formed by many species complexes that can be difficult to identify due to the similar shapes of species in this genus.Deudorix livia was described by Klug in 1834 based on the Lycaena genus, then the Virachola genus by Moore in 1881.Several studies about this genus were concerned ecology, economic harm, and control 1,8,[18][19][20][21] , but studies on the morphology of this pest are rare.Also, morphological studies were superficial, such as [22][23][24] .So, the current study aims to accurately identify the pomegranate butterfly type through a full description of morphological and genetic techniques.
Although morphometric identification is much better than general morphology of insect's species, using genetic tools became the most accurate method to differentiate between closely related species.Our results indicated that Deudorix livia was recorded for the first time in the GenBank for the partial nucleotide sequences of CO1 deposited under accession number MW463927.However, among the studied D. livia species, pairwise genetic distances ranged from 0.0114 to 0.0151.The most related species to our sample was Deudorix isocrates, although it differed morphologically from our sample 25 , where the genetic distance was 0.0114.Based on the finding of Kaleshkumar 26 who reported that closely related species have the lowest genetic distance, while the highest genetic distance refers to highly diverged cases.Some morphological features were studied for the first time, such as compound eyes focused on one scanned eye and interfacial hairs found between every three ommatidia.Meanwhile, most researchers focused on the general description of the eye as having dense white and dark scales on the borders, and others mentioned that it doesn't have ocelli 9,17,27 .Also, on the upper side of the male hindwing, there was one spotted patch in the subdiscal area and near the humeral angle (basal area).This feature is believed to be the female sexual pheromone receiving area during the mating process.So, it needs extensive future studies to identify such pheromones for using alternative pesticides to help control this pest.
The importance of real nomenclature for the pest is one of the most important steps for conducting appropriate control approach to reduce the population density of pomegranate butterfly below the economic injury level.

Conclusions
The findings indicate that both sexes' body scales are white on the ventral side and a mixture of dark and light grey on the dorsal side.Also, it contributes a similar wing venation.However, the average body length (BL) for male was 11.674 0.299 mm, while for female it was 11.458 1.001 mm.The mitochondrial CO1 gene from Deudorix livia was originally partially sequenced and deposited in GenBank under the accession number MW463927.The accurate identification of D. livia in pomegranate agroecosystems can help in designing appropriate (IPM) programs for this serious economic pest.This is in line with the global goals of sustainable development for environmental integrity and human health.

Figure 3 .
Figure 3.The body scales color of pomegranate butterfly adults: (a) female and (b) male.

Figure 4 .
Figure 4.The head capsule of a pomegranate butterfly: (a,b) Light micrograph, (c,d) SEM, red circle surrounds the interfacial hair in a corner between three facets.

Figure 5 .
Figure 5. Clavate type of pomegranate butterfly antenna by using light microscopy.

Figure 8 .
Figure 8.The wing upper side of a pomegranate butterfly (x = 8): (a) The female wing's upper side (a red circle surrounds two spots) and (b) The male wing's upper side (a black circle surrounds one spot), FW: fore wing and HW: hind wing.

Figure 9 .
Figure 9. Wing coloration of the lower side of pomegranate butterfly (x = 8): (a) The female wing's lower side, (b) The male wing's lower side (a red circle surrounds two spots female and male), FW: Fore Wing and HW: hind wing.

Figure 11 .
Figure 11.Abdomen description of pomegranate butterfly removed scales: (a) female and (b) male.

Figure 12 .
Figure 12.(a) Minimum-evolution phylogenetic tree among Deudorix livia with their related species, and the out-group using the (COX1) gene.(b) Neighbour Joining phylogenetic tree among Deudorix livia with their related species, and the out-group using (COX1) gene.

Table 1 .
Body length measurements from the head to the tip of the abdomen of the pomegranate butterfly.

Table 2 .
Wings length measurements fore wing length from humeral angle to apical angle and hind wing length from base to middle of apical margin in males and females for pomegranate butterfly.

Table 3 .
Accession numbers, nucleotide frequencies, A + T and C + G content of mitochondrial CO1 gene of Deudorix livia.

Table 5 .
Pairwise distances using the (COX1) gene among Deudorix livia with their related species, and the out-group.